P420: THE ROLE OF ACETYLCHOLINE IN THE REGULATION OF ACUTE MYELOID LEUKEMIA STEM CELLS
Authors: Michelle Buri, Xiaochi MA, Chantal Bachmann, Carina Stoll, Adrian Ochsenbein, Carsten Riether
Journal: HemaSphere (2023)

Background: Acute myeloid leukemia (AML) is the most frequent myeloid leukemia in adults and characterized by the accumulation highly proliferative, non-functioning myeloid blasts. The high relapse rate and therapy-resistance associated with the disease are mediated by a small population of persisting leukemic stem cells (LSCs) in the bone marrow (BM). Many LSC characteristics are, in part, mediated by interactions with the various cell types of the BM HSC niche, which they hijack to their advantage. Understanding these interactions is crucial to find prospective treatments targeting LSCs specifically. The neuronal input of the peripheral nervous system (PNS) to the niche is of emerging importance. Loss of sympathetic signals was shown to lead to premature ageing of the BM niche and cholinergic signals were linked to aberrant activation of hematopoietic stem cells. Aims: In this study, we investigated the effect of the parasympathetic part of the PNS, its main neurotransmitter acetylcholine (ACh) and the role of ACh producing non-neuronal niche cells and their importance for LSC maintenance and AML. Methods: Analysis of primary human/mouse LSCs/HSCs by RT-qPCR and FACS for the expression of cholinergic system markers (i.e. the different acetylcholine receptor subunits, choline acetyltransferase and acetylcholine esterases). Analysis of ACh in human/mouse serum with ELISA. CFU and replating assays of primary human/mouse LSCs and HSCs to determine the effect of ACh on stem cell function in vitro. The syngeneic MLL-AF9 mouse model in combination with subdiaphragmatic vagotomy to determine the contribution of vagus-nerve derived ACh on leukemia and LSCs. RNA sequencing to analyse the signaling cascades triggered by vagus-nerve derived Ach in LSCs. Treatment of MLL-AF9 mice with the nicotinic ACh receptor (AChR) antagonist hexamethonium to evaluate the potential of pharmacological AChR blockade in AML. Results: We found that ACh levels were significantly elevated in serum of newly diagnosed AML patients compared to age matched healthy donors. Higher ACh levels correlated with a worse survival in AML. Human and mouse HSCs as well as their respective LSCs expressed cholinergic system markers. Mouse and human LSCs responded to ACh in vitro manifesting in increased growth rates and increased colony formation in serial-replating experiments while neither mouse nor human HSCs displayed similar responses. The vagotomy mouse model revealed that the numbers of functional LSCs, but not healthy HSCs is reduced by lower peripheral concentrations of ACh. To further elucidate which intracellular mechanisms are primarily responsible for the effect of ACh, we sequenced the total RNA content from LSCs originating from vagotomised and sham operated mice. On a single gene level, different histone variants as well as the Bcl2 family member Bax were upregulated in LSCs from vagotomised mice while Notch 1, Jak 1 and NF-κB were downregulated. Gene ontology analysis revealed downregulation of typical immune cell signalling cascades and calcium ion binding in those LSCs, and upregulation of epigenetic mechanisms. The GSEA further confirmed these findings as the Ca2+, cAMP and lipid signalling pathway, the PI3K-Akt pathway, MAPK pathway and leukemic stem cell maintenance pathways were downregulated in LSCs derived from vagotomised MLL-AF9 mice. Lastly, pharmacological blockage of AChRs with hexamethonium in MLL-AF9 AML mice reduced LSC in vivo. Summary/Conclusion: We show that ACh promotes stemness of LSCs but not HSCs in mice and humans. Mechanistically this may be due to epigenetic modulation of central signaling cascades regulating stemness of LSCs such as the PI3K-Akt and MAPK pathway. Keywords: Bone marrow niche, AML